Crystallisation of a GLP-1 analogue

ABSTRACT

Crystals of glucagon-like peptide-1 (GLP-1) and GLP-1 analogues, and processes for preparation of crystals of GLP-1 and GLP-1 analogues.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/769,692filed on Jan. 25, 2001, now pending and claims priority under 35 U.S.C.119 of Danish application no. PA 2000 00156 filed Jan. 31, 2000; and ofU.S. provisional application No. 60/183,300 filed on Feb. 17, 2002; thecontents of which are fully incorporated herein by reference.

The present invention relates to novel crystals of GLP-1 and analoguesthereof, such as needle shaped crystals, and processes for thepreparation of crystals of GLP-1 and analogues thereof.

BACKGROUND

The hormones regulating insulin secretion belong to the so-calledenteroinsular axis, designating a group of hormones, released from thegastrointestinal mucosa in response to the presence and absorption ofnutrients in the gut, which promote an early and potentiated release ofinsulin. The enhancing effect on insulin secretion, the so-calledincretin effect, is probably essential for a normal glucose tolerance.Many of the gastrointestinal hormones, including gastrin and secretin(cholecystokinin is not insulinotropic in man), are insulinotropic, butthe only physiologically important ones, those that are responsible forthe incretin effect, are the glucose-dependent insulinotropicpolypeptide, GIP, and glucagon-like peptide-1 (GLP-1). Because of itsinsulinotropic effect, GIP, isolated in 1973 (1) immediately attractedconsiderable interest among diabetologists. However, numerousinvestigations carried out during the following years clearly indicatedthat a defective secretion of GIP was not involved in the pathogenesisof insulin-dependent diabetes mellitus (IDDM) or non-insulin-dependentdiabetes mellitus (NIDDM) (2). Furthermore, as an insulinotropichormone, GIP was found to be almost ineffective in NIDDM (2). The otherincretin hormone, GLP-1 is the most potent insulinotropic substanceknown (3). Unlike GIP, it is surprisingly effective in stimulatinginsulin secretion in NIDDM patients. In addition, and in contrast to theother insulinotropic hormones (perhaps with the exception of secretin)it also potently inhibits glucagon secretion. Because of these actionsit has pronounced blood glucose lowering effects particularly inpatients with NIDDM.

DESCRIPTION OF THE INVENTION

Human GLP-1 is a 37 amino acid residue peptide originating frompreproglucagon which is synthesised i.a. in the L-cells in the distalileum, in the pancreas and in the brain. Processing of preproglucagon togive GLP-1(7-36)amide, GLP-1(7-37) and GLP-2 occurs mainly in theL-cells. A simple system is used to describe fragments and analogues ofthis peptide. Thus, for example, Gly⁸-GLP-1 (7-37) designates a fragmentof GLP-1 formally derived from GLP-1 by deleting the amino acid residuesNos. 1 to 6 and substituting the naturally occurring amino acid residuein position 8 (Ala) by Gly. Similarly,Lys³⁴(N^(ε)-tetradecanoyl)-GLP-1(7-37) designates GLP-1(7-37) whereinthe ε-amino group of the Lys residue in position 34 has beentetradecanoylated.

GLP-1 and analogues thereof can be produced by a method which comprisesculturing or fermenting a host cell containing a DNA sequence encodingthe GLP-1 analogue and capable of expressing said analogue in a suitablenutrient medium under conditions permitting the expression of thepeptide, after which the resulting GLP-1 analogue is recovered from theculture or fermentation broth.

The implementation of a crystallisation step in the manufacturingprocess for the preparation of a GLP-1 analogue resulted in removal ofcoloured compounds (coloured impurities) from the fermentation broth,reduction of yeast host cell proteins, such as Saccharomyces cerevisiaeproteins (SCP) as well as removal of water, and low loss of the GLP-1analog from the mother liquor.

The GLP-1 analog was then re-dissolved and further purified byconventional High Performance Cation Exchange Chromatography (HP-CIEC)and Reverse Phase High Performance Liquid Chromatography (RP-HPLC)followed by a second crystallisation step at the pl of the GLP-1analogue. Hereafter, the analogue was acylated, e.g. as disclosed in WO98/08871, and the resulting solution containing mono-acylated GLP-1analogue was further purified by conventional RP-HPLC and finallyprecipitated at pl of the mono-acylated GLP-1 analogue.

Thus, the resulting crystals are an important and useful intermediateproduct in the manufacturing process for preparing a GLP-1 analogue andfor preparing a mono-acylated GLP-1 analogue. The resulting crystals ofthe GLP-1 analogue are also useful in the preparation of apharmaceutical composition, such as an injectable drug, comprising thecrystals and a pharmaceutically acceptable carrier.

Accordingly, the present invention relates to a process for producingcrystals of a GLP-1 analogue comprising:

-   a) preparing an aqueous solution comprising a GLP-1 analogue, a    salt, and an organic solvent, and-   b) isolating the crystals after formation.

The GLP-1 analogue to be crystallized in step a) is either substantiallypure or is impure. The purity can be measured by analytical HPLC and/orcapillary electrophoresis.

In step a) a buffer may optionally be added to said solution. Usually itis most convenient to add a buffer to the solution, such as any bufferincluding but not limited to: citrate buffers, phosphate buffers, trisbuffers, bis-Tris buffer, borate buffers, lactate buffers, glycyl glycinbuffers, arginine buffers, carbonate buffers, acetate buffers, glutamatebuffers, ammonium buffers, glycin buffers, alkylamine buffers,aminoethyl alcohol buffers, ethylenediamine buffers, tri-ethanol amine,imidazole buffers, pyridine buffers and barbiturate buffers and mixturesthereof. The concentration of buffer added is easily decided by theskilled practitioner using his common general knowledge, but willusually be in the range from 0 mM to 100 mM, such as 0.5 mM to 50 mM,e.g. 5-10 mM.

Further in step a) pH of the solution may be adjusted by means of anacid or base to keep it constant or it may vary, provided that pl of theGLP-1 analogue is not reached. Usually it is most convenient to adjustpH with an acid, e.g. HCl, if pH of the aqueous solution is above theisoelectric point (hereinafter pl) of the GLP-1 analogue, or with abase, e.g. NaOH, if pH of the aqueous solution is below the pl of theGLP-1 analogue. The pH of the solution is easily decided by the skilledpractitioner using his common general knowledge, but will usually bewithin a certain range from the pl of the GLP-1 analogue, such asbetween about pl−4 (id est, pl minus 4) and pl or between about pl andpl+4 (id est pl plus 4), depending on whether the aqueous solution ofthe GLP-1 analogue is below the pl or above the pl. The pH of theaqueous solution may be adjusted relatively far from the pl of the GLP-1analogue, such as at about pl−4 or pl+4 and then, optionally, by step orlinear gradient, be driven towards the pl until formation of crystalsoccur, in particular needle shaped crystals. In case of the GLP-1analogue being Arg³⁴GLP-1(7-37), having a pl of about 5.4, it ispreferred to prepare an aqueous solution of Arg³⁴GLP-1(7-37) above pl,preferably at about pH 8.5-9.5, and then adjust the pH to about pH 6-7with an acid, e.g. HCl. In one embodiment of the invention the pH isadjusted so that pl−4<pH<pl, preferably pl-2<pH<pl. In anotherembodiment of the invention the pH is adjusted so that pl<pH<pl+4,preferably pl<pH<pl+2.

Further in step a) an excipient may optionally be added that influencethe stability or solubility of the GLP-1 analogue.

In one embodiment of the invention the crystals are needle shapedcrystals of a GLP-1 analogue having a length of at least 0.5 μm. Inanother embodiment of the invention the crystals are needle shapedcrystals of a GLP-1 analogue having a length of at least 2 μm. In afurther embodiment of the invention the crystals are needle shapedcrystals of a GLP-1 analogue having a length of at least 8 μm. In afurther embodiment of the invention the crystals are needle shapedcrystals of a GLP-1 analogue having a length of 0.5 μm to 50 μm, such as2 μm to 50 μm, e.g. 2 μm to 30 μm. In a further embodiment of theinvention the crystals are needle shaped crystals of a GLP-1 analoguehaving a length of 8 μm to 50 μm.

In another embodiment of the invention the GLP-1 analogue to becrystallized has a purity of less than 98%, as measured by HPLC. In afurther embodiment of the invention the GLP-1 analogue to becrystallized has a purity of less than 95%, such as less than 90%, asmeasured by HPLC.

In another embodiment of the invention the GLP-1 analogue to becrystallized has a purity of more than 20%, such as more than 30%, asmeasured by HPLC. In a further embodiment of the invention the GLP-1analogue to be crystallized has a purity between 20% and 90%, such as apurity between 20% and 50%, as measured by HPLC.

In a further embodiment of the invention the GLP-1 analogue in theaqueous solution is present in a concentration of at least 0.5 mg/ml.

In a further embodiment of the invention the GLP-1 analogue in theaqueous solution is present in a concentration of from 0.5 mg/ml to 20mg/ml, such as 2-10 mg/ml.

In a further embodiment of the invention the GLP-1 analogue is selectedfrom non-synthetic GLP-1 analogues.

In a further embodiment of the invention the GLP-1 analogue is selectedfrom the Thr⁸, Met⁸, Gly⁸ and Val⁸ analogues of GLP-1(7-37) andGLP-1(7-36) amide, more preferred the Gly⁸ and Val⁸ analogues ofGLP-1(7-37) and GLP-1(7-36) amide, most preferred the Val⁸ analogues ofGLP-1(7-37) and GLP-1(7-36) amide.

In a further embodiment of the invention the GLP-1 analogue has theformula II: (SEQ ID: NO 1)   7   8   9    10 11  12   13 14  15  16 17His-Xaa-Xaa-Gly-Xaa-Phe-Thr-Xaa-Asp-Xaa-Xaa- 18   19  20  21 22   2324  25   26  27  28 Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Phe-29  30  31  32  33  34  35  36  37  38Ile-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa 39  40  41  42   43  44 45Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa (II)wherein

-   -   Xaa at position 8 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,        Asp, Met, or Lys,    -   Xaa at position 9 is Glu, Asp, or Lys,    -   Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu,        Asp, or Lys,    -   Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, lie, Val, Glu,        Asp, or Lys,    -   Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr,        Glu, Asp, or Lys,    -   Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu,        Asp, or Lys,    -   Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu,        Asp, or Lys,    -   Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, or Lys,    -   Xaa at position 20 is Leu, Ala, Gly, Ser, Thr, Leu, Ile, Val,        Glu, Asp, or Lys,    -   Xaa at position 21 is Glu, Asp, or Lys,    -   Xaa at position 22 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu,        Asp, or Lys,    -   Xaa at position 23 is Gin, Asn, Arg, Glu, Asp, or Lys,    -   Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg,        Glu, Asp, or Lys,    -   Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,        Asp, or Lys,    -   Xaa at position 26 is Lys, Arg, Gin, Glu, Asp, or His,    -   Xaa at position 27 is Glu, Asp, or Lys,    -   Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,        Asp, or Lys,    -   Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, or Lys,    -   Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu,        Asp, or Lys,    -   Xaa at position 33 is Val, Gly, Ala, Ser, Thr, Leu, Ile, Glu,        Asp, or Lys,    -   Xaa at position 34 is Lys, Arg, Glu, Asp, or His,    -   Xaa at position 35 is Gly, Ala, Ser, Thr, Leu; Ile, Val, Glu,        Asp, or Lys,    -   Xaa at position 36 is Arg, Lys, Glu, Asp, or His,    -   Xaa at position 37 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu,        Asp, or Lys, or is deleted,    -   Xaa at position 38 is Arg, Lys, Glu, Asp, or His, or is deleted,    -   Xaa at position 39 is Arg, Lys, Glu, Asp, or His, or is deleted,    -   Xaa at position 40 is Asp, Glu, or Lys, or is deleted,    -   Xaa at position 41 is Phe, Trp, Tyr, Glu, Asp, or Lys, or is        deleted,    -   Xaa at position 42 is Pro, Lys, Glu, or Asp, or is deleted,    -   Xaa at position 43 is Glu, Asp, or Lys, or is deleted,    -   Xaa at position 44 is Glu, Asp, or Lys, or is deleted, and    -   Xaa at position 45 is Val, Glu, Asp, or Lys, or is deleted, or

-   (a) a C-1-6-ester thereof, (b) amide, C-1-6-alkylamide, or    C-1-6-dialkylamide thereof and/or (c) a pharmaceutically acceptable    salt thereof, provided that    -   (i) when the amino acid at position 37, 38, 39, 40, 41, 42, 43        or 44 is deleted, then each amino acid downstream of the amino        acid is also deleted.

In a further embodiment of the GLP-1 analogue of formula II, the aminoacids at positions 37-45 are absent.

In another embodiment of the GLP-1 analogue of formula II, the aminoacids at positions 38-45 are absent.

In another embodiment of the GLP-1 analogue of formula II, the aminoacids at positions 39-45 are absent.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 8 is Ala, Gly, Ser, Thr, Met, or Val.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 8 is Gly, Thr, Met, or Val.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 8 is Val.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 9 is Glu.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 11 is Thr.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 14 is Ser.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 16 is Val.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 17 is Ser.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 18 is Ser, Lys, Glu, or Asp.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 19 is Tyr, Lys, Glu, or Asp.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 20 is Leu, Lys, Glu, or Asp.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 21 is Glu, Lys, or Asp.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 22 is Gly, Glu, Asp, or Lys.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 23 is Gin, Glu, Asp, or Lys.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 24 is Ala, Glu, Asp, or Lys.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 25 is Ala, Glu, Asp, or Lys.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 26 is Lys, Glu, Asp, or Arg.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 27 is Glu, Asp, or Lys.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 30 is Ala, Glu, Asp, or Lys.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 31 is Trp, Glu, Asp, or Lys.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 32 is Leu, Glu, Asp, or Lys.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 33 is Val, Glu, Asp, or Lys.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 34 is Lys, Arg, Glu, or Asp.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 35 is Gly, Glu, Asp, or Lys.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 36 is Arg, Lys, Glu, or Asp.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 37 is Gly, Glu, Asp, or Lys.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 38 is Arg, or Lys, or is deleted.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 39 is deleted.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 40 is deleted.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 41 is deleted.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 42 is deleted.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 43 is deleted.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 44 is deleted.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 45 is deleted.

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 26 is Arg, each of Xaa at positions 37-45 is deleted, and eachof the other Xaa is the amino acid in native GLP-1 (7-36).

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 26 is Arg, each of Xaa at positions 38-45 is deleted, and eachof the other Xaa is the amino acid in native GLP-1 (7-37).

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 26 is Arg, each of Xaa at positions 39-45 is deleted, and eachof the other Xaa is the amino acid in native GLP-1 (7-38).

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 34 is Arg, each of Xaa at positions 37-45 is deleted, and eachof the other Xaa is the amino acid in native GLP-1 (7-36).

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 34 is Arg, each of Xaa at positions 38-45 is deleted, and eachof the other Xaa is the amino acid in native GLP-1 (7-37).

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 34 is Arg, each of Xaa at positions 39-45 is deleted, and eachof the other Xaa is the amino acid in native GLP-1 (7-38).

In another embodiment of the GLP-1 analogue of formula II, Xaa atpositions 26 and 34 is Arg, Xaa at position 36 is Lys, each of Xaa atpositions 37-45 is deleted, and each of the other Xaa is the amino acidin native GLP-1 (7-36).

In another embodiment of the GLP-1 analogue of formula II, Xaa atpositions 26 and 34 is Arg, Xaa at position 36 is Lys, each of Xaa atpositions 38-45 is deleted, and each of the other Xaa is the amino acidin native GLP-1 (7-37).

In another embodiment of the GLP-1 analogue of formula II, Xaa atpositions 26 and 34 is Arg, Xaa at position 36 is Lys, each of Xaa atpositions 39-45 is deleted, and each of the other Xaa is the amino acidin native GLP-1 (7-38).

In another embodiment of the GLP-1 analogue of formula II, Xaa atpositions 26 and 34 is Arg, Xaa at position 38 is Lys, each of Xaa atpositions 39-45 is deleted, and each of the other Xaa is the amino acidin native GLP-1 (7-38).

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 8 is Thr, Ser, Gly, or Val, Xaa at position 37 is Glu, Xaa atposition 36 is Lys, each of Xaa at positions 38-45 is deleted, and eachof the other Xaa is the amino acid in native GLP-1 (7-37).

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 8 is Thr, Ser, Gly, or Val, Xaa at position 37 is Glu, Xaa atposition 36 is Lys, each of Xaa at positions 39-45 is deleted, and eachof the other Xaa is the amino acid in native GLP-1 (7-38).

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 8 is Thr, Ser, Gly or Val, Xaa at position 37 is Glu, Xaa atposition 38 is Lys, each of Xaa at positions 39-45 is deleted, and eachof the other Xaa is the amino acid in native GLP-1 (7-38).

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 18, 23 or 27 is Lys, and Xaa at positions 26 and 34 is Arg,each of Xaa at positions 37-45 is deleted, and each of the other Xaa isthe amino acid in native GLP-1 (7-36).

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 18, 23 or 27 is Lys, and Xaa at positions 26 and 34 is Arg,each of Xaa at positions 38-45 is deleted, and each of the other Xaa isthe amino acid in native GLP-1(7-37).

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 18, 23 or 27 is Lys, and Xaa at positions 26 and 34 is Arg,each of Xaa at positions 39-45 is deleted, and each of the other Xaa isthe amino acid in native GLP-1 (7-38).

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 8 is Thr, Ser, Gly, or Val, Xaa at position 18, 23 or 27 isLys, and Xaa at position 26 and 34 is Arg, each of Xaa at positions37-45 is deleted, and each of the other Xaa is the amino acid in nativeGLP-1(7-36).

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 8 is Thr, Ser, Gly, or Val, Xaa at position 18, 23 or 27 isLys, and Xaa at position 26 and 34 is Arg, each of Xaa at positions38-45 is deleted, and each of the other Xaa is the amino acid in nativeGLP-1(7-37).

In another embodiment of the GLP-1 analogue of formula II, Xaa atposition 8 is Thr, Ser, Gly, or Val, Xaa at position 18, 23 or 27 isLys, and Xaa at position 26 and 34 is Arg, each of Xaa at positions39-45 is deleted, and each of the other Xaa is the amino acid in nativeGLP-1(7-38).

Such GLP-1 analogues of formula II includes, but is not limited to,Arg²⁶-GLP-1(7-37); Arg³⁴-GLP-1(7-37); Lys³⁶-GLP-1(7-37);Arg^(26,34)Lys³⁶-GLP-1(7-37); Arg^(26,34)Lys³⁸GLP-1(7-38);Arg^(26,34)Lys³⁹-GLP-1(7-39); Arg^(26,34)Lys⁴⁰-GLP-1(7-40);Arg²⁶Lys³⁶-GLP-1(7-37); Arg 34Lys³⁶-GLP-1(7-37); Arg²⁶Lys³⁹-GLP-1(7-39);Arg³⁴Lys⁴⁰-GLP-1(7-40); Arg^(26,34)Lys^(36,39)-GLP-1(7-39);Arg^(26,34)Lys^(36,40)-GLP-1(7-40); Gly⁸Arg²⁶-GLP-1(7-37);Gly⁸Arg³⁴-GLP-1(7-37); Val⁸-GLP-1(7-37); Thr⁸-GLP-1(7-37);Gly⁸-GLP-1(7-37); Met⁸-GLP-1(7-37); Gly³Lys³⁶-GLP-1(7-37);Gly⁸Arg^(26,34)Lys³⁶-GLP-1(7-37); Gly⁸Arg^(26,34)Lys³⁹-GLP-1(7-39);Gly⁸Arg^(26,34)Lys⁴⁰-GLP-1(7-40); Gly⁸Arg²⁶Lys³⁶-GLP-1(7-37);Gly⁸Arg³⁴Lys³⁶-GLP-1(7-37); Gly⁸Arg²⁶Lys³⁹-GLP-1(7-39);Gly⁸Arg³⁴Lys⁴⁰-GLP-1(7-40); Gly⁸Arg^(26,34)Lys^(36,39)-GLP-1(7-39);Gly⁸Arg^(26,34)Lys^(36,40)-GLP-1(7-40); Arg ^(26,34)Lys³⁸GLP-1(7-38);Arg^(26,34)Lys³⁹GLP-1(7-39); Arg^(26,34)Lys⁴⁰GLP-1(7-40);Arg^(26,34)Lys⁴¹GLP-1(7-41); Arg^(26,34)Lys⁴²GLP-1(7-42);Arg^(26,34)Lys⁴³GLP-1(7-43); Arg²⁶Lys⁴⁴GLP-1(7-44);Arg^(26,34)Lys⁴⁵GLP-1(7-45); Arg^(26,34)Lys³⁸GLP-1(1-38);Arg^(26,34)Lys³⁹GLP-1(1-39); Arg^(26,34)Lys⁴⁰GLP-1(1-40);Arg^(26,34)Lys⁴¹GLP-1(1-41); Arg^(26,34)Lys⁴²GLP-1(1-42);Arg^(26,34)Lys⁴³GLP-1(1-43); Arg^(26,34)Lys⁴⁴GLP-1(1-44);Arg^(26,34)Lys⁴⁵GLP-1(1-45); Arg^(26,34)Lys³⁸GLP-1(2-38); Arg^(26,34)Lys³⁹GLP-1(2-39); Arg^(26,34)Lys⁴⁰GLP-1(2-40);Arg^(26,34)Lys⁴¹GLP-1(2-41); Arg^(26,34) Lys⁴²GLP-1(2-42);Arg^(26,34)Lys⁴³GLP-1(2-43); Arg^(26,34)Lys⁴⁴GLP-1(2-44);Arg^(26,34)Lys⁴⁵GLP-1(2-45); Arg^(26,34)Lys³⁸GLP-1(3-38);Arg^(26,34)Lys³⁹GLP-1(3-39); Arg^(26,34)Lys⁴⁰GLP-1(3-40);Arg^(26,34)Lys⁴¹GLP-1(3-41); Arg^(26,34)Lys⁴²GLP-1(3-42);Arg^(26,34)Lys⁴³GLP-1(3-43); Arg^(26,34)Lys⁴⁴GLP-1(3-44);Arg^(26,34)Lys⁴⁵GLP-1(345); Arg^(26,34)Lys³⁸GLP-1(4-38);Arg^(26,34)Lys³⁹GLP-1(4-39); Arg^(26,34)Lys⁴⁰GLP-1(4-40);Arg^(26,34)Lys⁴¹GLP-1(4-41); Arg^(26,34)Lys⁴²GLP-1(4-42);Arg^(26,34)Lys⁴³GLP-1(4-43); Arg^(26,34)Lys⁴⁴GLP-1(4-44);Arg^(26,34)Lys⁴⁵GLP-1(4-45); Arg^(26,34)Lys³⁸GLP-1(5-38);Arg^(26,34)Lys³⁹GLP-1(5-39); Arg^(26,34)Lys⁴⁰GLP-1(5-40);Arg^(26,34)Lys⁴¹GLP-1(5-41); Arg^(26,34)Lys⁴²GLP-1(5-42);Arg^(26,34)Lys⁴³GLP-1(5-43); Arg^(26,34)Lys⁴⁴GLP-1(5-44);Arg^(26,34)Lys⁴⁵GLP-1(5-45); Arg^(26,34)Lys³⁸GLP-1(6-38);Arg^(26,34)Lys³⁹GLP-1(6-39); Arg^(26,34)Lys⁴⁰GLP-1(6-40);Arg^(26,34)Lys⁴¹GLP-1(6-41); Arg^(26,34)Lys⁴²GLP-1(6-42);Arg^(26,34)Lys⁴³GLP-1(6-43); Arg^(26,34)Lys⁴⁴GLP-1(6-44); Arg^(26,34)Lys ⁴⁵GLP-1(6-45); Arg²⁶Lys³⁸GLP-1(1-38); Arg³⁴Lys³⁸GLP-1(1-38);Arg^(26,34)Lys^(36,38)GLP-1(1-38); Arg²⁶Lys³⁸GLP-1(7-38);Arg³⁴Lys³⁸GLP-1(7-38); Arg^(26,34)Lys^(36,38)GLP-1(7-38);Arg^(26,34)Lys³⁸GLP-1(7-38); Arg²⁶Lys³⁹GLP-1(1-39);Arg³⁴Lys³⁹GLP-1(1-39); Arg^(26,34)Lys^(36,39)GLP-1(1-39);Arg²⁶Lys³⁹GLP-1(7-39); Arg³⁴Lys³⁹GLP-1(7-39) andArg^(26,34)Lys^(36,39)GLP-1(7-39). Each one of these specific GLP-1analogues constitutes an alternative embodiment of the invention.

In a further embodiment of the invention the GLP-1 analogue has theformula IIIA-HN-GLP-1(8-B)-X  (III)wherein

-   A is:    wherein R¹, R² and R³ are independently H, lower alkyl having 1 to 6    carbon atoms, optionally substituted phenyl, NH₂, NH—CO-(lower    alkyl), —OH, lower alkoxy having 1 to 6 carbon atoms, halogen, SO₂—    (lower alkyl) or CF₃, said phenyl is optionally substituted with at    least one group selected from NH₂, —OH, lower alkyl or lower alkoxy    having 1-6 carbon atoms, halogen, SO₂-(lower alkyl), NH—CO-(lower    alkyl) or CF₃, or R¹ and R² may together form a bond;-   Y is a five or six membered ring system selected from the group    consisting of:    wherein Z is N, O or S, said ring system is optionally substituted    with one or more functional groups selected from the group    consisting of NH₂, NO₂, OH, C₁₋₄ alkyl, C₁₋₄ alkoxy, halogen (Cl,    Br, F, I), CF₃ and aryl;-   B is an integer in the range of 35-45; and-   X is —OH, —NH₂, or a C₁₋₄ alkyl amide or C₁₋₄ dialkyl amide group;    or an analogue thereof.

Such GLP-1 analogues of formula III includes, but is not limited to

-   Arg²⁶-GLP-1(7-37); Arg³⁴-GLP-1(7-37); Lys³⁶-GLP-1(7-37);    Arg^(26,34)Lys³⁶-GLP-1(7-37); Arg^(26,34)Lys³⁸GLP-1(7-38);    Arg^(26,34)Lys³⁹-GLP-1 (7-39); Arg^(26,34)Lys⁴⁰-GLP-1 (7-40);    Arg²⁶Lys³⁶-GLP-1(7-37); Arg³⁴Lys³⁶-GLP-1(7-37);    Arg²⁶Lys³⁹-GLP-1(7-39); Arg³⁴Lys⁴⁰-GLP-1(7-40);    Arg^(26,34)Lys^(36,39)-GLP-1 (7-39); Arg^(26,34)Lys³⁶40-GLP-1(7-40);    Gly⁸Arg²⁶-GLP-1(7-37); Gly⁸Arg³⁴-GLP-1(7-37); Gly⁸Lys³⁶-GLP-1    (7-37); Gly⁸Arg^(26,34)Lys³⁶-GLP-1(7-37);    Gly⁸Arg^(26,34)Lys³⁹-GLP-1(7-39); Gly⁸Arg^(26,34)Lys⁴⁰-GLP-1 (7-40);    Gly⁸Arg²⁶Lys³⁶-GLP-1(7-37); Gly⁸Arg³⁴Lys³⁶-GLP-1(7-37);    Gly⁸Arg²⁶Lys³⁹-GLP-1(7-39); Gly⁸Arg³⁴Lys⁴⁰-GLP-1 (740);    Gly⁸Arg^(26,34)Lys^(36,39)-GLP-1(7-39); or-   Gly⁸Arg^(26,34)Lys³⁶40-GLP-1(7-40). Each one of these specific GLP-1    analogues constitutes an alternative embodiment of the invention.

In a further embodiment of the invention the GLP-1 analogue has theformula IVA-GLP-1(19-B)-X  (IV)wherein

-   -   A is a peptide comprising the amino acid residues of GLP-1(8-18)        or a fragment thereof;    -   B is an integer in the range of 35-45; and    -   X is H, —NH₂, or a C₁₋₆ alkyl amide or C₁₋₆ dialkyl amide group;        or an analogue thereof.

In an embodiment of the GLP-1 analogue of formula IV, A is a peptideselected from the group consisting of GLP-1 (8-18), GLP-1(9-18),GLP-1(10-18), GLP-1(11-18), GLP-1(12-18), GLP-1(13-18), GLP-1(14-18),GLP-1(15-18), GLP-1(16-18), GLP-1(17-18) and GLP-1(18). Preferably, A isGLP-1(8-18), GLP-1(9-18), GLP-1(10-18), GLP-1(11-18) or GLP-1(12-18),and B is 36, 37 or 38. Most preferably, A is GLP-1(8-18).

In a further embodiment of the GLP-1 analogue of formula IV, B is 35,36, 37, 38, 39, 40, 41, 42, 43 or 44. In a more preferred embodiment, Bis 36. In another more preferred embodiment. B is 37. In another morepreferred embodiment, B is 38.

Such GLP-1 analogues of formula IV includes, but is not limited to

-   Arg²⁶-GLP-1 (8-37); Arg³⁴-GLP-1 (8-37); Lys³⁶-GLP-1 (8-37);    Arg^(26,34)Lys³⁶-GLP-1 (8-37); Arg^(26,34)Lys³³GLP-1(838);    Arg^(26,34)Lys³⁹-GLP-1 (8-39); Arg^(26,34)Lys⁴⁰-GLP-1 (8-40);    Arg²⁶Lys³⁶-GLP-1(8-37); Arg³⁴Lys³⁶-GLP-1(8-37);    Arg²⁶Lys³⁹-GLP-1(8-39); Arg³⁴Lys⁴⁰-GLP-1(8-40);    Arg^(26,34)Lys^(36,39)-GLP-1 (8-39);    Arg^(26,34)Lys^(36,40)GLP-1(840); Gly⁸Arg²⁶-GLP-1(8-37);    Gly³Arg³⁴-GLP-1(8-37); Gly⁸Lys³⁶-GLP-1(8-37);    Gly⁸Arg^(26,34)Lys³⁶-GLP-1(8-37); Gly⁸Arg ^(26,34)Lys³⁹-GLP-1(8-39);    Gly⁸Arg^(26,34)Lys⁴⁰-GLP-1 (8-40); Gly⁸Arg²⁶Lys³⁶-GLP-1(8-37);    Gly⁸Arg³⁴Lys³⁶-GLP-1(8-37); Gly⁸Arg²⁶Lys³⁹-GLP-1(8-39);    Gly⁸Arg³⁴Lys⁴⁰-GLP-1(8-40); Gly⁸Arg^(26,34)Lys^(36,39)-GLP-1(8-39);    or-   Gly⁸Arg^(26,34)Lys^(36,40)-GLP-1(8-40). Each one of these specific    GLP-1 analogues constitutes an alternative embodiment of the    invention.

In a further embodiment of the invention the GLP-1 analogue isArg²⁶GLP-1(7-37).

In a further embodiment of the invention the GLP-1 analogue isArg³⁴GLP-1 (7-37).

In a further embodiment of the invention the salt is present in aconcentration of at least 25 mM. In a further embodiment of theinvention the salt is present in a concentration of from 25 mM to 800mM, such as 100 to 200 mM. The salt may be added to the aqueous solutioncomprising the GLP-1 analogue all at once or as a step or lineargradient.

In a further embodiment of the invention the salt is selected frominorganic salts. Such inorganic salts, includes but is not limited tochlorides, bromides, fluorides, iodides, sulphates or nitrates withammonium, alkaline metals or earth alkaline metals, or mixtures thereof,e.g. NaCl, KCl, NH₄Cl, CaCl₂, sodium sulphate, ammonium nitrate,potassium sulphate, ammonium sulphate, or mixtures thereof.

In a further embodiment of the invention the salt is selected fromorganic salts. Such organic salts, includes but is not limited toacetates, citrates or tartrates with ammonium, alkaline metals or earthalkaline metals, or mixtures thereof, e.g. sodium acetate, potassiumacetate, ammonium acetate, sodium citrate, potassium citrate, potassiumtartrate, ammonium citrate, calcium acetate or mixtures thereof.

In a further embodiment of the invention the organic solvent is presentin a concentration of at least 0.5% (vol/vol). In a further embodimentof the invention the organic solvent is present in a concentration offrom 0.5 to 50% (vol/vol), such as 1 to 15% (vol/vol). The organicsolvent may be added to the aqueous solution comprising the GLP-1analogue all at once or as a step or linear gradient.

In a further embodiment of the invention the organic solvent is selectedfrom C₁₋₆ alkanol, C₁₋₆alkenol, C₁₋₆alkynol, urea, guanidine,C₁₋₆-alkanoic acid, ketone, DMSO, C₂₋₆-glycol, C₃₋₇-polyalcoholincluding sugars, or mixtures thereof. Each of these solventsconstitutes an individual embodiment of the invention.

In a further embodiment of the invention the organic solvent is selectedfrom C₁₋₆ alkanol, ketone or C₃₋₇-polyalcohol including sugars.

In a further embodiment of the invention the organic solvent is selectedfrom methanol, ethanol, n-propanol, allyl alcohol, n-butanol,n-pentanol, n-hexanol, 2-propanol, tert-butyl alcohol, 1,2-ethanediol,1,2-propanediol, 2-methyl-2,4-pentanediol, glycerol, methylethyl ketoneor acetone. Each of these solvents constitutes an individual embodimentof the invention. The organic solvent is preferably selected fromethanol, glycerol or acetone.

After preparation of the solution comprising a GLP-1 analogue, it isnormally placed at ambient temperature, and the crystals will start toform after a while. After formation the crystals are isolated from themother liquor. The temperature of the solution is easily decided by theskilled practitioner using his common general knowledge, and he maydecide to place the solution at a constant temperature, or optionallyplace the solution at one temperature and then by step or lineargradient move the temperatur to a lower temperature.

In a further embodiment of the invention the solution is placed at atemperature from about −10° C. to +40° C. In further embodiments of theinvention the solution is placed at a temperature from −5° C. to 40° C.,−2° C. to 40° C., −1° C. to 40° C., 4° C. to 37° C., or 20 to 25° C.Each of these ranges constitutes an individual embodiment of theinvention.

Formation of the crystals may start after 10-60 minutes although it maytake a shorther or longer period of time. After formation of thecrystals they may be isolated from the mother liquor by filtration,decantation, centrifugation or other means known to the skilledpractitioner.

The present invention also relates to crystals, preferably needle shapedcrystals, of a GLP-1 analogue optainable by the process comprising:

-   a) preparing an aqueous solution comprising a GLP-1 analogue, a    salt, and an organic solvent, and-   b) isolation of the crystals after formation.

The present invention also relates to needle shaped crystals of a GLP-1analogue having a length of at least 0.5 μm. In another embodiment ofthe invention the crystals are needle shaped crystals of a GLP-1analogue having a length of at least 2 μm. In a further embodiment ofthe invention the crystals are needle shaped crystals of a GLP-1analogue having a length of 0.5 μm to 50 μm, such as 2 μm to 50 μm, e.g.2 μm to 30 μm. In a further embodiment of the invention the crystals areneedle shaped crystals of a GLP-1 analogue having a length of at least 8μm. In a further embodiment of the invention the crystals are needleshaped crystals of a GLP-1 analogue having a length of 8 μm to 50 μm.

In a further aspect the present invention relates to a pharmaceuticalcomposition comprising crystals, preferably needle shaped crystals, of aGLP-1 analogue together with a pharmaceutically acceptable carrier orexcipient.

The GLP-1 analogues can be produced by a method which comprisesculturing or fermenting a host cell containing a DNA sequence encodingthe GLP-1 analogue and capable of expressing said analogue in a suitablenutrient medium under conditions permitting the expression of thepeptide, after which the resulting GLP-1 analogue is recovered from theculture or fermentation broth. Hereinafter, culturing will be used tocover both culturing and fermenting and the like.

The medium used to culture the cells may be any conventional mediumsuitable for growing the host cells, such as minimal or complex mediacontaining appropriate supplements. Suitable media are available fromcommercial suppliers or may be prepared according to published recipes(e.g. in catalogues of the American Type Culture Collection). The GLP-1analogue produced by the cells may then be recovered from the culturemedium by conventional procedures including, optionally lysis of cells,separating the host cells from the medium by centrifugation orfiltration, precipitating the proteinaceous components of thesupernatant or filtrate by means of a salt, e.g. ammonium sulphate,purification by conventional purification techniques, such aschromatographic techniques, if necessary, purification by ion exchangechromatography according to the present invention, and subsequently,subjecting to analytical tests, e.g. PAGE, IEF, if necessary, subjectingto further purification, if necessary, and isolation of the pure GLP-1analogue.

The DNA sequence encoding the GLP-1 analogue may suitably be of genomicor cDNA origin, for instance obtained by preparing a genomic or cDNAlibrary and screening for DNA sequences coding for all or part of theGLP-1 analogue by hybridisation using synthetic oligonucleotide probesin accordance with standard techniques (see, for example, Sambrook, J,Fritsch, E F and Maniatis, T, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequenceencoding the GLP-1 analogue may also be prepared synthetically byestablished standard methods, e.g. the phosphoamidite method describedby Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, orthe method described by Matthes et al., EMBO Journal 3 (1984), 801-805.The DNA sequence may also be prepared by polymerase chain reaction usingspecific primers, for instance as described in U.S. Pat. No. 4,683,202or Saiki et al., Science 239 (1988), 487-491.

The DNA sequence may be inserted into any vector which may convenientlybe subjected to recombinant DNA procedures, and the choice of vectorwill often depend on the host cell into which it is to be introduced.Thus, the vector may be an autonomously replicating vector, i.e. avector which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g. a plasmid.Alternatively, the vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequenceencoding the GLP-1 analogue is operably linked to additional segmentsrequired for transcription of the DNA, such as a promoter. The promotermay be any DNA sequence which shows transcriptional activity in the hostcell of choice and may be derived from genes encoding proteins eitherhomologous or heterologous to the host cell. Examples of suitablepromoters for directing the transcription of the DNA encoding the GLP-1analogue in a variety of host cells are well known in the art, cf. forinstance Sambrook et al., supra.

The DNA sequence encoding the GLP-1 analogue may also, if necessary, beoperably connected to a suitable terminator, polyadenylation signals,transcriptional enhancer sequences, and translational enhancersequences. The recombinant vector may further comprise a DNA sequenceenabling the vector to replicate in the host cell in question.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell or one whichconfers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin,chloramphenicol, neomycin, hygromycin or methotrexate.

To direct a GLP-1 analogue into the secretory pathway of the host cells,a secretory signal sequence (also known as a leader sequence, preprosequence or pre sequence) may be provided in the recombinant vector. Thesecretory signal sequence is joined to the DNA sequence encoding theGLP-1 analogue in the correct reading frame. Secretory signal sequencesare commonly positioned 5′ to the DNA sequence encoding the GLP-1analogue. The secretory signal sequence may be that normally associatedwith the GLP-1 analogue or may be from a gene encoding another secretedprotein.

The procedures used to ligate the DNA sequences coding for the GLP-1analogue, the promoter and optionally the terminator and/or secretorysignal sequence, respectively, and to insert them into suitable vectorscontaining the information necessary for replication, are well known topersons skilled in the art (cf., for instance, Sambrook et al., supra).

The host cell into which the DNA sequence or the recombinant vector isintroduced may be any cell which is capable of producing the GLP-1analogue and includes bacteria, vira, e.g. baculo virus, yeast, fungi,insect cells and higher eukaryotic cells. Examples of suitable hostcells well known and used in the art are, without limitation, E coli,Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.

Some of the GLP-1 analogue, can be produced according to conventionalorganic peptide synthetic chemistry. The resulting synthetic mixture maythen be chemically modified, e.g. by alkylation, acylation, esterformation or amide formation or the like, and purified, or purified asit is and then modified chemically.

Usually, the fermentation broth comprising the GLP-1 analogue will alsocontain amino acids, small peptides, large peptides, unrelated proteins,reactants, cell debris, host cell proteins, endotoxins, and/or viradepending on whether recombinant DNA techniques and/or chemicalmodification techniques have been used or whether organic peptidesynthetic chemistry techniques have been used.

Thus, any method, such as an industrial method, for producing a GLP-1analogue, which includes a crystallization step according to the presentinvention is also an aspect of the present application.

Accordingly, the present invention relates in a further aspect to amethod for producing a GLP-1 analogue or a GLP-1 analogue whereto isattached a lipophilic substituent comprising:

-   a) expressing the GLP-1 analogue in a host cell, such as yeast,-   b) precipitating the analogue at its pl,-   c) preparing an aqueous solution comprising the GLP-1 analogue, a    salt, and an organic solvent, and-   d) isolation of the crystals after formation, and-   e) further purification, optionally followed by repetition of steps    b to e, to isolation of crystals of the GLP-1 analogue, and-   f) optionally acylation of the GLP-1 analogue, optionally followed    by purification.

In a further aspect the present invention relates to a process forproducing crystals of a GLP-1 analogue comprising:

-   a) preparing an aqueous solution comprising a GLP-1 analogue, and a    salt, and-   b) isolation of the crystals after formation.

Another invention relates to a process for producing crystals of a GLP-1analogue comprising:

-   a) preparing an aqueous solution comprising a GLP-1 analogue, and a    salt, and-   b) isolation of the crystals after formation. Anyone of the above    embodiments is also intended to represent embodiments of this    invention where possible.

The term “an organic solvent”, as used herein, is intended to includeany organic solvent which do not denature the GLP-1 analogue. Theorganic solvent includes but is not limited to C₁₋₆-alkanol,C₁₋₆-alkenol or C₁₋₆-alkynol, urea, guanidine, or C₁₋₆alkanoic acid,such as acetic acid, ketone, such as acetone, dimethylsulphoxide (DMSO),polymeric solvents, C₂₋₆-glycol, C₃₋₇-polyalcohol including sugars, ormixtures thereof.

The term “C₁₋₆alkanol”, “C₁₋₆alkenol” or “C₁₋₆alkynol”, as used herein,alone or in combination is intended to include those C₁₋₆alkane,C₁₋₆alkene or C₁₋₆alkyne groups of the designated length in either alinear or branched or cyclic configuration whereto is linked a hydroxyl(—OH) (cf. Morrison & Boyd, Organic Chemistry, 4^(th) ed). Examples oflinear alcohols are methanol, ethanol, n-propanol, allyl alcohol,n-butanol, n-pentanol and n-hexanol. Examples of branched alcohols are2-propanol and tert-butyl alcohol. Examples of cyclic alcohols are cyclopropyl alcohol and 2-cyclohexen-1-ol.

The term “C₁₋₆alkanoic acid”, as used herein, is intended to include agroup of the formula R′COOH wherein R′ is H or C₁₋₅alkyl, such asacetic, propionic, butyric, α-methylbutyric, or valeric acid (cf.Morrison & Boyd, Organic Chemistry, 4^(th) ed).

The term “C₁₋₁₂-alkyl”, as used herein, is intended to include abranched or straight alkyl group having from one to 12 carbon atoms.Typical C₁₋₁₂-alkyl groups are C₁₋₅-alkyl groups which include, but arenot limited to, methyl, ethyl, n-propyl, iso-propyl, butyl, iso-butyl,sec-butyl, tert-butyl, pentyl, iso-pentyl, and the like (cf. Morrison &Boyd, Organic Chemistry, 4^(th) ed).

The term “C₂₋₆-glycol”, as used herein, is intended to include aC₂₋₆-alkane containing two hydroxyl groups on different carbon atomswhich may be adjacent or not. A typical C₂₋₆-glycol include, but is notlimited to 1,2-ethanediol, 1,2-propanediol, or 2-methyl-2,4-pentanediol(cf. Morrison & Boyd, Organic Chemistry, 4^(th) ed).

The term “C₂₋₄-alkane”, as used herein, is intended to include abranched or straight alkane group having from two to six carbon atoms.Typical C₂₋₆-alkane groups include, but are not limited to ethane,propane, iso-propane, butane, iso-butane, pentane, hexane and the like(cf. Morrison & Boyd, Organic Chemistry, 4^(th) ed).

The term “C₃₋₇-polyalcohol including sugars”, as used herein, isintended to include a group of the formula HOCH₂(CHOH)_(n)CH₂OH whereinn is an integer from 1-5, and monosaccharides such as glycerol, mannose,glucose (cf. Morrison & Boyd, Organic Chemistry, 4^(th) ed).

The term “a GLP-1 analogue”, as used herein, is intended to designateGLP-1 (7-37), GLP-1 (7-36) amide as well as analogues, fragments,homologues, and derivatives thereof, which are capable of being producedby conventional recombinant DNA techniques as well as conventionalsynthetic methods. Such GLP-1 analogues include but are not limited tonative glucagon-like peptide-1, for instance such peptide fragmentswhich comprises GLP-1 (7-37) and functional derivatives thereof asdisclosed in WO 87/06941; such peptide fragments which comprise GLP-1(7-36) and functional derivatives thereof as disclosed in WO 90/11296;such analogues of the active GLP-1 peptides 7-34, 7-35, 7-36, and 7-37as disclosed in WO 91/11457; such N-terminal truncated fragments ofGLP-1 as disclosed in EP 0699686-A2; such GLP-1 analogues andderivatives that include an N-terminal imidazole group as disclosed inEP 0708179-A2; and such exendins as disclosed in WO 9746584 and U.S.Pat. No. 5,424,286.

The term “exendins”, as used herein, is intended to designate exendin aswell as analogs, derivatives, and fragments thereof, e.g. exendin-3 and-4. Examples of exendin as well as analogs, derivatives, and fragmentsthereof to be included within the present invention are those disclosedin WO 9746584 and U.S. Pat. No. 5,424,286. U.S. Pat. No. 5,424,286describes a method for stimulating insulin release with exendinpolypeptide(s). The exendin polypeptides disclosed includeHGEGTFTSDLSKQMEEEAVRLFIEWLKNGGX; wherein X═P or Y, andHX1X2GTFITSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS; wherein X1×2=SD (exendin-3)or GE (exendin-4)). The exendin-3 and -4 and fragments are useful intreatment of diabetes mellitus (types I or II) and prevention ofhyperglycaemia. They normalise hyperglycaemia through glucose-dependent,insulin-independent and insulin-dependent mechanisms. Exendin-4 isspecific for exendin receptors, i.e. it does not interact withvasoactive intestinal peptide receptors. WO 9746584 describes truncatedversions of exendin peptide(s) for treating diabetes. The disclosedpeptides increase secretion and biosynthesis of insulin, but reducethose of glucagon. The truncated peptides can be made more economicallythan full length versions.

The term “crystals” as used herein, is intended to designate crystals ofany shape, such as single needle shaped crystals (which is the same asneedle-like crystals), single irregular shaped crystals, single oblongcrystals as well as clusters of two or more crystals and mixturesthereof (cf. “Preparation and analysis of protein crystals” by A.McPherson).

The term “non-synthetic GLP-1 analogues” as used herein, is intended todesignate a GLP-1 analogue which comprises only naturally occurringamino acid residues and is capable of being produced by recombinant DNAtechniques or expressed by organisms, e.g. microorganisms.

The term “ketone” as used herein, is intended to designate a compound ofthe formula R¹—CO—R² wherein R¹ and R² are independently of each otherselected from C₁₋₁₂-alkyl, preferably C₁₋₅-alkyl (cf. Morrison & Boyd,Organic Chemistry, 4^(th) ed).

The term “polymeric solvents” as used herein, is intended to comprisepoly(acrylic acid), carboxymethylcellulose, poly(ethylene glycol),poly(propylene glycol), poly(vinyl alcohol), poly(vinylpyrrolidone) andthe like (cf. “Preparation and analysis of protein crystals” by A.McPherson).

The term “analogues” as used herein, is intended to designate a peptidewherein one or more amino acid residues of the parent peptide have beensubstituted by another amino acid residue and/or wherein one or moreamino acid residues of the parent peptide have been deleted and/orwherein one or more amino acid residues have been added to the parentpeptide. Such addition can take place either at the N-terminal end or atthe C-terminal end of the parent peptide or both.

The term “derivatives” as used herein, is intended to designate apeptide in which one or more of the amino acid residues of the parentpeptide have been chemically modified, e.g. by alkylation, acylation,ester formation or amide formation or the like.

The term “a salt” as used herein, is intended to include any organic orinorganic salt, including but not limited to NaCl, KCl, NH₄Cl, CaCl₂,sodium acetate, potassium acetate, ammonium acetate, sodium citrate,potassium citrate, ammonium citrate, sodium sulphate, potassiumsulphate, ammonium sulphate, calcium acetate or mixtures thereof (cf.Remington's Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co.,Easton, Pa., 1990, or Remington: The Science and Practice of Pharmacy,19th Edition (1995), or handbooks from Amersham-Pharmacia Biotech).

The term “a buffer” as used herein, is intended to include any bufferincluding but not limited to: citrate buffers, phosphate buffers, trisbuffers, bis-Tris buffer, borate buffers, lactate buffers, glycyl glycinbuffers, arginine buffers, carbonate buffers, acetate buffers, glutamatebuffers, ammonium buffers, glycin buffers, alkylamine buffers,aminoethyl alcohol buffers, ethylenediamine buffers, tri-ethanol amine,imidazole buffers, pyridine buffers and barbiturate buffers and mixturesthereof (cf. Remington's Pharmaceutical Sciences, Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, or Remington: The Science andPractice of Pharmacy, 19th Edition (1995), or handbooks fromAmersham-Pharmacia Biotech).

The present invention is further illustrated by the following exampleswhich, however, are not to be construed as limiting the scope ofprotection. The features disclosed in the foregoing description and inthe following examples may, both separately and in any combinationthereof, be material for realising the invention in diverse formsthereof.

EXPERIMENTAL PART

Crystallisation of Arg³⁴GLP1₍₇₋₃₇₎ in the Manufacturing Process forPreparing a Mono Acylated GLP-1 Analogue

Arg³⁴GLP1₍₇₋₃₇₎ was expressed in yeast, that is Saccharomyces cerevisiae(Sacch. cerevisiae), by conventional recombinant DNA technology. Thefermentation broth was purified by a conventional reverse phase capturestep followed by a first precipitation step (precipitate (A)) at theiso-electric point (pl) of Arg³⁴GLP1₍₇₋₃₇₎. Arg³⁴GLP1₍₇₋₃₇₎ was thenredissolved and further purified by conventional High Performance CationExchange Chromatography (HP-CIEC) and Reverse Phase High PerformanceLiquid Chromatography (RP-HPLC) followed by a second precipitation step(precipitate (B)) at the pl of Arg³⁴GLP1₍₇₋₃₇₎.

Then Arg³⁴GLP1₍₇₋₃₇₎ was acylated, e.g. as disclosed in WO 98/08871, andthe resulting solution containing mono-acylated Arg³⁴GLP1₍₇₋₃₇₎ wasfurther purified by conventional RP-HPLC and finally precipitated at plof mono-acylated Arg³⁴GLP1₍₇₋₃₇₎.

The implementation of a crystallisation step in the manufacturingprocess for the preparation of mono-acylated Arg³⁴GLP1₍₇₋₃₇₎ results inremoval of coloured compounds from the fermentation broth, reduction ofSaccharomyces cerevisiae proteins (host cell proteins) (SCP) as well asremoval of water, and low loss of Arg³⁴GLP1₍₇₋₃₇₎ from the motherliquor.

Crystallisation of Arg³⁴GLP1₍₇₋₃₇₎ from precipitate (A) and precipitate(B) has been performed. An overview of the experiments is given in thefollowing section along with a description of the crystallisationprocedure.

General Procedure:

The precipitate (A) from the first precipitation step (or theprecipitate (B) from the second precipitation step) was suspended inwater and pH was adjusted to pH 8.5-9.5 with NaOH by which theprecipitate dissolved. The Arg³⁴GLP1₍₇₋₃₇₎ concentration (referred to asGLP1 conc. in the tables) of the stock solution was measured byanalytical RP-HPLC (Analytical procedure 427-AF006.D02: Purity andConcentration of GLP-1 (Inger Bastholm, 1999)). Then salt, organicsolvent and buffer compound was added to the desired concentration andthe solution was adjusted to the specified pH with HCl, then gentlyswirled and placed at the specified temperature (scale: 3-5 ml).Formation of crystals started to occur after 10-60 minutes and after16-18 hr the crystal morphology was studied in microscope (MicroscopeBX50 from Olympus). For quantification a portion of the mother liquorwas removed and centrifuged. The Arg³⁴GLP1₍₇₋₃₇₎ content was measured byanalytical RP-HPLC. The loss by crystallisation was calculated bydivision of the supernatant content by the content in the startingmaterial.

N.A.=Not Assessed Example GLP1 Loss precipitate A conc. Salt SolventBuffer pH Temp. Crystal morphology/size [%]  1 3.4 mg/ml  75 Mm NaClNone 6 mM bis-Tris 6.5 20-25° C. Amorphous precipitate. 45.6  2 ″  75 MmNaCl   5% ethanol 6 mM bis-Tris 6.5 20-25° C. Irregular shaped. 13.5  3″  75 mM NaCl  10% ethanol 6 mM bis-Tris 6.5 20-25° C. Irregular shaped.12.4  4 ″  75 mM NaCl  15% ethanol 6 mM bis-Tris 6.5 20-25° C. Amorphousprecipitate. 13.0  5 ″ 150 mM NaCl None 6 mM bis-Tris 6.5 20-25° C.Irregular shaped. 13.4  6 ″ 150 mM NaCl   5% ethanol 6 mM bis-Tris 6.520-25° C. Irregular shaped, few needle 11.2 shaped (ca. 5 μm).  7 ″ 150mM NaCl  10% ethanol 6 mM bis-Tris 6.5 20-25° C. Needle shaped (5-15μm). 9.1  8 ″ 150 mM NaCl  15% ethanol 6 mM bis-Tris 6.5 20-25° C.Amorphous precipitation. 11.3  9 3.4 mg/ml 150 mM NaCl  10% ethanol 6 mMbis-Tris 6.6 20-25° C. Needle shaped. 5.7 10 ″ 200 mM NaCl  10% ethanol6 mM bis-Tris 6.6 20-25° C. Needle shaped. 5.9 11 ″ 250 mM NaCl  10%ethanol 6 mM bis-Tris 6.5 20-25° C. Needle shaped, clusters. 5.7 12 ″300 mM NaCl  10% ethanol 6 mM bis-Tris 6.6 20-25° C. Needle shaped,clusters. 5.1 13 5.6 mg/ml 150 mM NaCl 2.5% acetone 5 mM bis-Tris 6.520-25° C. Small, irregular shaped. 8.4 14 ″ 150 mM NaCl   5% acetone 5mM bis-Tris 6.5 20-25° C. Small, oblong crystals 8.2 (ca. 5 μm). 15 ″150 mM NaCl  10% acetone 5 mM bis-Tris 6.5 20-25° C. Irregular shaped,many 8.9 needle shaped (7-9 μm). 16 ″ 150 mM NaCl 2.5% glycerol 5 mMbis-Tris 6.5 20-25° C. Small, irregular shaped 8.6 (1-2 μm). 17 ″ 150 mMNaCl   5% glycerol 5 mM bis-Tris 6.5 20-25° C. Small, irregular shaped.9.5 (2-3 μm). 18 ″ 150 mM NaCl  10% glycerol 5 mM bis-Tris 6.5 20-25° C.Small, irregular shaped 8.0 (1-2 μm). Example GLP1 Loss Precipitate Bconc. Salt Solvent Buffer pH Temp. Crystal morphology/size [%] 19   4mg/ml  75 mM NaCl   5% ethanol 5 mM bis-Tris 6.5 20-25° C. Needleshaped. 4.0 20 ″  75 mM NaCl   5% ethanol 5 mM bis-Tris 6.5   4° C.Needle shaped. 6.5 21 ″  75 mM NaCl  10% ethanol 5 mM bis-Tris 6.520-25° C. Needle shaped. 4.3 22 ″  75 mM NaCl  10% ethanol 5 mM bis-Tris6.5   4° C. Needle shaped. 4.5 23 ″  75 mM NaCl  15% ethanol 5 mMbis-Tris 6.5 20-25° C. Needle shaped. 5.3 24 ″  75 mM NaCl  15% ethanol5 mM bis-Tris 6.5   4° C. Needle shaped. 6.0 25 ″ 100 mM NaCl None 5 mMbis-Tris 6.5 20-25° C. Needle shaped. 3.8 26 ″ 100 mM NaCl None 5 mMbis-Tris 6.5   4° C. Amorphous. 7.0 27 ″ 100 mM NaCl   5% ethanol 5 mMbis-Tris 6.5 20-25° C. Needle shaped. N.A. 28 ″ 100 mM NaCl   5% ethanol5 mM bis-Tris 6.5   4° C. Irregular shaped. 6.0 29 ″ 100 mM NaCl  10%ethanol 5 mM bis-Tris 6.5 20-25° C. Needle shaped. 3.3 30 ″ 100 mM NaCl 10% ethanol 5 mM bis-Tris 6.5   4° C. Needle shaped. 4.0 31 ″ 100 mMNaCl  15% ethanol 5 mM bis-Tris 6.5 20-25° C. Needle shaped. 4.0 32 ″100 mM NaCl  15% ethanol 5 mM bis-Tris 6.5   4° C. Needle shaped,clusters. 1.3 33 ″ 200 mM NaCl None 5 mM bis-Tris 6.5 20-25° C.Granular, few needle 2.8 shaped. 34 ″ 200 mM NaCl None 5 mM bis-Tris 6.5  4° C. Amorphous precipitation. 2.8 35 ″ 200 mM NaCl  10% ethanol 5 mMbis-Tris 6.5 20-25° C. Needle shaped. 2.8 36 ″ 200 mM NaCl  10% ethanol5 mM bis-Tris 6.5   4° C. Amorphous, few needle 2.0 shaped. 37 ″ 300 mMNaCl None 5 mM bis-Tris 6.5 20-25° C. Granular, few needle 2.3 shaped.38 ″ 300 mM NaCl None 5 mM bis-Tris 6.5   4° C. Amorphous. 2.0 39 ″ 300mM NaCl  10% ethanol 5 mM bis-Tris 6.5 20-25° C. Needle shaped. 2.5 40 ″300 mM NaCl  10% ethanol 5 mM bis-Tris 6.5   4° C. Amorphousprecipitation, 2.0 few needle shaped. 41 3.3 mg/ml 100 mM NaCl   2%ethanol 5 mM bis-Tris 6.5 20-25° C. Needle shaped (ca. 7 μm). N.A. 423.2 mg/ml 100 mM NaCl   5% ethanol 5 mM bis-Tris 6.5 20-25° C. Needleshaped (7-11 μm). 4.9 43 3.2 mg/ml 100 mM NaCl   7% ethanol 5 mMbis-Tris 6.5 20-25° C. Needle shaped (7-11 μm). 4.9 44 3.1 mg/ml 100 mMNaCl  10% ethanol 5 mM bis-Tris 6.5 20-25° C. Needle shaped (7-9 μm).5.5 45 2.9 mg/ml 100 mM NaCl  15% ethanol 5 mM bis-Tris 6.5 20-25° C.Needle shaped (7-9 μm) N.A. clusters. 46 3.2 mg/ml 200 mM NaCl   2%ethanol 5 mM bis-Tris 6.5 20-25° C. Needle shaped (ca. 7 μm). N.A. 473.1 mg/ml 200 mM NaCl   5% ethanol 5 mM bis-Tris 6.5 20-25° C. Needleshaped (7-11 μm). 4.7 48 3.0 mg/ml 200 mM NaCl   7% ethanol 5 mMbis-Tris 6.5 20-25° C. Needle shaped (7-11 μm). 5.2 49 2.9 mg/ml 200 mMNaCl  10% ethanol 5 mM bis-Tris 6.5 20-25° C. Needle shaped (7-11 μm).6.2 50 2.8 mg/ml 200 mM NaCl  15% ethanol 5 mM bis-Tris 6.5 20-25° C.Needle shaped (7-9 μm), N.A. clusters. 51 2.6 mg/ml  75 mM NaCl   7%ethanol 5 mM bis-Tris 6.5 20-25° C. Needle shaped (5-6 μm). 5.8 52 2.3mg/ml 300 mM NaCl   7% ethanol 5 mM bis-Tris 6.5 20-25° C. Needle shaped(9-12 μm). 5.8 53 3.4 mg/ml 100 mM KCl   7% ethanol 5 mM bis-Tris 6.520-25° C. Needle shaped (4-9 μm). 4.8 54 3.2 mg/ml 200 mM KCl   7%ethanol 5 mM bis-Tris 6.5 20-25° C. Needle shaped (4-9 μm), 4.7 55 3.4mg/ml 100 mM (NH₄)₂SO₄   7% ethanol 5 mM bis-Tris 6.5 20-25° C. Needleshaped (7-9 μm). 5.1 56 3.2 mg/ml 200 mM (NH₄)₂SO₄   7% ethanol 5 mMbis-Tris 6.5 20-25° C. Needle shaped (4-7 μm). 5.4 57 0.5 mg/ml 200 mMNaCl   7% ethanol 5 mM bis-Tris 6.5 20-25° C. Few needle shaped. 22.5 580.6 mg/ml 200 mM NaCl   7% ethanol 5 mM bis-Tris 6.5 20-25° C. Fewneedle shaped. 18.8 59 1.0 mg/ml 200 mM NaCl   7% ethanol 5 mM bis-Tris6.5 20-25° C. Few needle shaped. 11.7 60 1.4 mg/ml 200 mM NaCl   7%ethanol 5 mM bis-Tris 6.5 20-25° C. Needle shaped. 8.8 61 2.0 mg/ml 200mM NaCl   7% ethanol 5 mM bis-Tris 6.5 20-25° C. Needle shaped. 4.5 623.0 mg/ml 200 mM NaCl   7% ethanol 5 mM bis-Tris 6.5 20-25° C. Needleshaped. 4.7 63 5.0 mg/ml 200 mM NaCl   7% ethanol 5 mM bis-Tris 6.520-25° C. Irregular shaped. 3.0 64 5.8 mg/ml 200 mM NaCl   7% ethanol 5mM bis-Tris 6.5 20-25° C. Amorphous precipitation. 3.2 65 3.2 mg/ml 200mM NaCl   7% ethanol 5 mM bis-Tris 6.0 20-25° C. Needle shaped. 2.1 66 ″200 mM NaCl   7% ethanol 5 mM bis-Tris 6.2 20-25° C. Needle shaped. 2.867 ″ 200 mM NaCl   7% ethanol 5 mM bis-Tris 6.6 20-25° C. Needle shaped.4.0 68 ″ 200 mM NaCl   7% ethanol 5 mM bis-Tris 7.0 20-25° C. Needleshaped. 5.5 69 ″ 200 mM NaCl   7% ethanol 5 mM bis-Tris 7.2 20-25° C.Needle shaped. 6.6

1. A process for producing crystals of a GLP-1 analogue comprising: a)preparing an aqueous solution comprising a GLP-1 analogue, a salt, andan organic solvent, and b) isolation of the crystals after formation. 2.The process according to claim 1 wherein in step a) adjusting pH topl−4<pH<pl, or to pl<pH<pl+4, wherein pl is the isoelectric point of theGLP-1 analogue.
 3. The process according to claim 1 wherein the crystalsare needle shaped crystals of a GLP-1 analogue.
 4. The process accordingto claim 1 wherein the crystals has a length of at least 0.5 μm.
 5. Theprocess according to claim 1 wherein the GLP-1 analogue in the aqueoussolution has a purity of less than 95%, as measured by HPLC.
 6. Theprocess according to claim 1 wherein the GLP-1 analogue in the aqueoussolution is present in a concentration of at least 0.5 mg/ml.
 7. Theprocess according to claim 1 wherein the GLP-1 analogue is selected fromnon-synthetic GLP-1 analogues.
 8. The process according to claim 1wherein the GLP-1 analogue is Arg³⁴GLP-1 (7-37) or Arg²⁶GLP-1 (7-37). 9.The process according to claim 1 wherein the salt is present in aconcentration of at least 25 mM.
 10. The process according to claim 1wherein the organic solvent is present in a concentration of from 0.5 to50% (vol/vol).
 11. The process according to claim 1 wherein the organicsolvent is selected from C₁₋₆alkanol, C₁₆-alkenol, C₁₋₆alkynol, urea,guanidine, C₁₋₆alkanoic acid, ketone, DMSO, C₂₋₄-glycol,C₃₋₇-polyalcohol including sugars, or mixtures thereof.
 12. A method forproducing a GLP-1 analogue or a GLP-1 analogue whereto is attached alipophilic substituent comprising the steps: a) preparing an aqueoussolution comprising a GLP-1 analogue, a salt, and an organic solvent,and b) isolation of the crystals after formation.
 13. Crystals of aGLP-1 analogue optainable by the method according to claim
 12. 14.Needle shaped crystals of a GLP-1 analogue.
 15. A pharmaceuticalcomposition comprising needle shaped crystals of a GLP-1 analogue and apharmaceutically acceptable carrier.